High activity mutants of butyrylcholinesterase for cocaine hydrolysis

ABSTRACT

Butyrylcholinesterase (BChE) polypeptide variants of the presently-disclosed subject matter have enhanced catalytic efficiency for (−)-cocaine, as compared to wild-type BChE. Pharmaceutical compositions of the presently-disclosed subject matter include a BChE polypeptide variant having an enhanced catalytic efficiency for (−)-cocaine. A method of the presently-disclosed subject matter for treating a cocaine-induced condition includes administering to an individual an effective amount of a BChE polypeptide variant, as disclosed herein, to lower blood cocaine concentration.

RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application Ser.No. 61/443,932 filed Feb. 17, 2011, and is a division of U.S. patentapplication Ser. No. 13/399,406 filed Feb. 17, 2012. The entiredisclosures contained in U.S. Provisional Application Ser. No.61/443,932 and U.S. application Ser. No. 13/399,406 are incorporatedherein by this reference.

GOVERNMENT INTEREST

Subject matter described herein was made with government support underGrant Number R01DA013930 awarded by the National Institute on Drug Abuse(NIDA) of the National Institutes of Health (NIH). The government hascertain rights in the described subject matter.

TECHNICAL FIELD

The presently-disclosed subject matter relates to butyrylcholinesterasevariant polypeptides, and in particular, butyrylcholinesterase mutantshaving amino acid substitutions.

INTRODUCTION

Cocaine abuse is a major medical and public health problem thatcontinues to defy treatment. The disastrous medical and socialconsequences of cocaine addiction, such as violent crime, loss inindividual productivity, illness, and death, have made the developmentof an effective pharmacological treatment a high priority. However,cocaine mediates its reinforcing and toxic effects by blockingneurotransmitter reuptake and the classical pharmacodynamic approach hasfailed to yield small-molecule receptor/transporter antagonists due tothe difficulties inherent in blocking a blocker. An alternative topharmacodynamic approach is to interfere with the delivery of cocaine toits receptors/transporters and accelerate its metabolism in the body.

The dominant pathway for cocaine metabolism in primates isbutyrylcholinesterase (BChE)-catalyzed hydrolysis at the benzoyl estergroup (Scheme 1).

Only 5% of the cocaine is deactivated through oxidation by the livermicrosomal cytochrome P450 system. Cocaine hydrolysis at benzoyl estergroup yields ecgonine methyl ester, whereas the oxidation producesnorcocaine. The metabolite ecgonine methyl ester is a biologicallyinactive metabolite, whereas the metabolite norcocaine is hepatotoxicand a local anesthetic. In human beings, more than 90% of cocaine ismetabolized by BChE. BChE is synthesized in the liver and widelydistributed in the body, including plasma, brain, and lung. Extensiveexperimental studies in animals and humans demonstrate that enhancementof BChE activity by administration of exogenous enzyme substantiallydecreases cocaine half-life.

Enhancement of cocaine metabolism by administration of BChE has beenrecognized to be a promising pharmacokinetic approach for treatment ofcocaine abuse and dependence. However, the catalytic activity of thisplasma enzyme is three orders-of-magnitude lower against the naturallyoccurring (−)-cocaine than that against the biologically inactive(+)-cocaine enantiomer. (+)-cocaine can be cleared from plasma inseconds and prior to partitioning into the central nervous system (CNS),whereas (−)-cocaine has a plasma half-life of approximately 45-90minutes (for a relatively low dose of cocaine), long enough formanifestation of the CNS effects which peak in minutes. Under theoverdose condition, BChE is saturated with (−)-cocaine and, thus, theplasma half-life of (−)-cocaine will be longer. Hence, BChE mutants withhigh activity against (−)-cocaine are highly desired for use in humans.Although some BChE mutants with increased catalytic activity overwild-type BChE have previously been generated, there exists a need formutant BChE with even higher catalytic activity.

SUMMARY

The presently-disclosed subject matter meets some or all of theabove-identified needs, as will become evident to those of ordinaryskill in the art after a study of information provided in this document.

This Summary describes several embodiments of the presently-disclosedsubject matter, and in many cases lists variations and permutations ofthese embodiments. This Summary is merely exemplary of the numerous andvaried embodiments. Mention of one or more representative features of agiven embodiment is likewise exemplary. Such an embodiment can typicallyexist with or without the feature(s) mentioned; likewise, those featurescan be applied to other embodiments of the presently-disclosed subjectmatter, whether listed in this Summary or not. To avoid excessiverepetition, this Summary does not list or suggest all possiblecombinations of such features.

The presently-disclosed subject matter includes butyrylcholinesterase(BChE) polypeptide variants. In some embodiments the amino acid sequenceof the BChE polypeptide variant includes an amino acid sequence selectedfrom the group consisting of: SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, and 46 as setforth herein. The presently-disclosed subject matter further includesnucleic acid molecules that encodes a BChE polypeptide variant,including the nucleic acid sequence selected from the group consistingof SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29,31, 33, 35, 37, 39, 41, 43, and 45.

The presently-disclosed subject matter further includes a pharmaceuticalcomposition that includes a butyrylcholinesterase polypeptide variantand a suitable pharmaceutical carrier.

The presently-disclosed subject matter further includes a method oftreating a cocaine-induced condition, which includes administering to anindividual an effective amount of BChE polypeptide variant or functionalfragment thereof, or a pharmaceutical composition comprising a BChEpolypeptide variant or functional fragment thereof, as described herein,to lower blood cocaine concentration. In some embodiments, the BChEpolypeptide variant exhibits a one-hundred-fold or more increase incocaine hydrolysis catalytic efficiency compared to wild-typebutyrylcholinesterase.

The presently-disclosed subject matter further includes a method oftreating a cocaine-induced condition, which includes administering to anindividual an effective amount of a BChE nucleotide variant, i.e., anucleotide molecule encoding a BChE polypeptide variant or functionalfragment thereof as disclosed herein.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 is a nucleotide sequence encoding a butyrylcholinesterase(BChE) polypeptide variant of SEQ ID NO: 2;

SEQ ID NO: 2 is an amino acid sequence encoding a BChE polypeptidevariant having the following amino acid substitutions, as compared towild type BChE: A199S, F227S, S287G, A328W, and Y332G;

SEQ ID NO: 3 is a nucleotide sequence encoding a butyrylcholinesterase(BChE) polypeptide variant of SEQ ID NO: 4;

SEQ ID NO: 4 is an amino acid sequence encoding a BChE polypeptidevariant having the following amino acid substitutions, as compared towild type BChE: A199S, F227T, S287G, A328W, and Y332G;

SEQ ID NO: 5 is a nucleotide sequence encoding a butyrylcholinesterase(BChE) polypeptide variant of SEQ ID NO: 6;

SEQ ID NO: 6 is an amino acid sequence encoding a BChE polypeptidevariant having the following amino acid substitutions, as compared towild type BChE: A199S, F227M, S287G, A328W, and Y332G;

SEQ ID NO: 7 is a nucleotide sequence encoding a butyrylcholinesterase(BChE) polypeptide variant of SEQ ID NO: 8;

SEQ ID NO: 8 is an amino acid sequence encoding a BChE polypeptidevariant having the following amino acid substitutions, as compared towild type BChE: A199S, F227C, S287G, A328W, and Y332G;

SEQ ID NO: 9 is a nucleotide sequence encoding a butyrylcholinesterase(BChE) polypeptide variant of SEQ ID NO: 10;

SEQ ID NO: 10 is an amino acid sequence encoding a BChE polypeptidevariant having the following amino acid substitutions, as compared towild type BChE: A199S, F227A, P285N, S287G, A328W, and Y332G;

SEQ ID NO: 11 is a nucleotide sequence encoding a butyrylcholinesterase(BChE) polypeptide variant of SEQ ID NO: 12;

SEQ ID NO: 12 is an amino acid sequence encoding a BChE polypeptidevariant having the following amino acid substitutions, as compared towild type BChE: A199S, F227P, P285A, S287G, A328W, and Y332G;

SEQ ID NO: 13 is a nucleotide sequence encoding a butyrylcholinesterase(BChE) polypeptide variant of SEQ ID NO: 14;

SEQ ID NO: 14 is an amino acid sequence encoding a BChE polypeptidevariant having the following amino acid substitutions, as compared towild type BChE: A199S, F227S, P285Q, S287G, A328W, and Y332G;

SEQ ID NO: 15 is a nucleotide sequence encoding a butyrylcholinesterase(BChE) polypeptide variant of SEQ ID NO: 16;

SEQ ID NO: 16 is an amino acid sequence encoding a BChE polypeptidevariant having the following amino acid substitutions, as compared towild type BChE: A199S, F227S, P285S, S287G, A328W, and Y332G;

SEQ ID NO: 17 is a nucleotide sequence encoding a butyrylcholinesterase(BChE) polypeptide variant of SEQ ID NO: 18;

SEQ ID NO: 18 is an amino acid sequence encoding a BChE polypeptidevariant having the following amino acid substitutions, as compared towild type BChE: A199S, F227S, P285G, S287G, A328W, and Y332G;

SEQ ID NO: 19 is a nucleotide sequence encoding a butyrylcholinesterase(BChE) polypeptide variant of SEQ ID NO: 20;

SEQ ID NO: 20 is an amino acid sequence encoding a BChE polypeptidevariant having the following amino acid substitutions, as compared towild type BChE: A199S, F227P, P285S, L286M, S287G, A328W, and Y332G;

SEQ ID NO: 21 is a nucleotide sequence encoding a butyrylcholinesterase(BChE) polypeptide variant of SEQ ID NO: 22;

SEQ ID NO: 22 is an amino acid sequence encoding a BChE polypeptidevariant having the following amino acid substitutions, as compared towild type BChE: A199S, F227A, P285S, S287G, A328W, and E441D;

SEQ ID NO: 23 is a nucleotide sequence encoding a butyrylcholinesterase(BChE) polypeptide variant of SEQ ID NO: 24;

SEQ ID NO: 24 is an amino acid sequence encoding a BChE polypeptidevariant having the following amino acid substitutions, as compared towild type BChE: A199S, F227A, P285A, S287G, A328W, and E441D;

SEQ ID NO: 25 is a nucleotide sequence encoding a butyrylcholinesterase(BChE) polypeptide variant of SEQ ID NO: 26;

SEQ ID NO: 26 is an amino acid sequence encoding a BChE polypeptidevariant having the following amino acid substitutions, as compared towild type BChE: A199S, F227P, L286M, S287G, A328W, and Y332G;

SEQ ID NO: 27 is a nucleotide sequence encoding a butyrylcholinesterase(BChE) polypeptide variant of SEQ ID NO: 28;

SEQ ID NO: 28 is an amino acid sequence encoding a BChE polypeptidevariant having the following amino acid substitutions, as compared towild type BChE: A199S, F227G, P285A, S287G, A328W, and Y332G;

SEQ ID NO: 29 is a nucleotide sequence encoding a butyrylcholinesterase(BChE) polypeptide variant of SEQ ID NO: 30;

SEQ ID NO: 30 is an amino acid sequence encoding a BChE polypeptidevariant having the following amino acid substitutions, as compared towild type BChE: A199S, F227G, P285G, S287G, A328W, and Y332G;

SEQ ID NO: 31 is a nucleotide sequence encoding a butyrylcholinesterase(BChE) polypeptide variant of SEQ ID NO: 32;

SEQ ID NO: 32 is an amino acid sequence encoding a BChE polypeptidevariant having the following amino acid substitutions, as compared towild type BChE: A199S, F227G, P285Q, S287G, A328W, and Y332G;

SEQ ID NO: 33 is a nucleotide sequence encoding a butyrylcholinesterase(BChE) polypeptide variant of SEQ ID NO: 34;

SEQ ID NO: 34 is an amino acid sequence encoding a BChE polypeptidevariant having the following amino acid substitutions, as compared towild type BChE: A199S, F227G, P285S, S287G, A328W, and Y332G;

SEQ ID NO: 35 is a nucleotide sequence encoding a butyrylcholinesterase(BChE) polypeptide variant of SEQ ID NO: 36;

SEQ ID NO: 36 is an amino acid sequence encoding a BChE polypeptidevariant having the following amino acid substitutions, as compared towild type BChE: A199S, F227A, P285E, S287G, A328W, and Y332G;

SEQ ID NO: 37 is a nucleotide sequence encoding a butyrylcholinesterase(BChE) polypeptide variant of SEQ ID NO: 38;

SEQ ID NO: 38 is an amino acid sequence encoding a BChE polypeptidevariant having the following amino acid substitutions, as compared towild type BChE: A199S, F227P, P285N, S287G, A328W, and Y332G;

SEQ ID NO: 39 is a nucleotide sequence encoding a butyrylcholinesterase(BChE) polypeptide variant of SEQ ID NO: 40;

SEQ ID NO: 40 is an amino acid sequence encoding a BChE polypeptidevariant having the following amino acid substitutions, as compared towild type BChE: A199S, F227S, P285A, S287G, A328W, and Y332G;

SEQ ID NO: 41 is a nucleotide sequence encoding a butyrylcholinesterase(BChE) polypeptide variant of SEQ ID NO: 42;

SEQ ID NO: 42 is an amino acid sequence encoding a BChE polypeptidevariant having the following amino acid substitutions, as compared towild type BChE: A199S, F227S, P285N, S287G, A328W, and Y332G;

SEQ ID NO: 43 is a nucleotide sequence encoding a butyrylcholinesterase(BChE) polypeptide variant of SEQ ID NO: 44;

SEQ ID NO: 44 is an amino acid sequence encoding a BChE polypeptidevariant having the following amino acid substitutions, as compared towild type BChE: A199S, F227S, L286M, S287G, A328W, and Y332G;

SEQ ID NO: 45 is a nucleotide sequence encoding a butyrylcholinesterase(BChE) polypeptide variant of SEQ ID NO: 46;

SEQ ID NO: 46 is an amino acid sequence encoding a BChE polypeptidevariant having the following amino acid substitutions, as compared towild type BChE: A199S, F227G, L286M, S287G, A328W, and Y332G;

DESCRIPTION OF EXEMPLARY EMBODIMENTS

The details of one or more embodiments of the presently-disclosedsubject matter are set forth in this document. Modifications toembodiments described in this document, and other embodiments, will beevident to those of ordinary skill in the art after a study of theinformation provided in this document. The information provided in thisdocument, and particularly the specific details of the describedexemplary embodiments, is provided primarily for clearness ofunderstanding and no unnecessary limitations are to be understoodtherefrom. In case of conflict, the specification of this document,including definitions, will control.

The presently-disclosed subject matter includes butyrylcholinesterase(BChE) polypeptide variants. The BChE polypeptide variants disclosedherein each have enhanced catalytic efficiency for (−)-cocaine, ascompared to wild-type BChE. The presently-disclosed subject matterfurther includes a pharmaceutical composition including abutyrylcholinesterase polypeptide variant, as described herein, and asuitable pharmaceutical carrier. The presently-disclosed subject matterfurther includes a method of treating a cocaine-induced conditioncomprising administering to an individual an effective amount of abutyrylcholinesterase polypeptide variant of functional fragment thereof, or a BChE nucleotide variant, i.e., a nucleotide molecule encoding aBChE polypeptide variant or functional fragment thereof, as disclosedherein, to lower blood cocaine concentration. A cocaine-inducedcondition includes conditions resulting from the administration/use ofcocaine, including, for example, treatment for an addiction to cocaine.For example, a polypeptide or nucleotide, as described herein, could beadministered prior to the use of cocaine as part of an addictiontreatment strategy.

In some embodiments, the BChE polypeptide variant is selected from aBChE polypeptide variants set forth in Table 1. Table 1 also includesthe SEQ ID NOs associated with the identified BChE polypeptide variants,as well as a summary of the approximate fold increase in catalyticefficiency against (−)-cocaine for the identified BChE polypeptidevariants, as compared to wild-type BChE.

TABLE 1 BChE Polypeptide Variants and Associated SEQ ID NOs Amino AcidSubstitution N.A. A.A. Var- SEQ SEQ iant ID ID No. 199 227 285 286 287328 332 441 C.E.^(a) NO: NO: 1 A199S F227S — — S287G A328W Y332G — 2750 1  2 2 A199S F227T — — S287G A328W Y332G — 1780  3  4 3 A199S F227M — —S287G A328W Y332G — 1310  5  6 4 A199S F227C — — S287G A328W Y332G —1290  7  8 5 A199S F227A P285N — S287G A328W Y332G — 2790  9 10 6 A199SF227P P285A — S287G A328W Y332G — 2810 11 12 7 A199S F227S P285Q — S287GA328W Y332G — 4260 13 14 8 A199S F227S P285S — S287G A328W Y332G — 243015 16 9 A199S F227S P285G — S287G A328W Y332G — 2480 17 18 10 A199SF227P P285S L286M S287G A328W Y332G — 2710 19 20 11 A199S F227A P285S —S287G A328W — E441D 2280 21 22 12 A199S F227A P285A — S287G A328W —E441D 1980 23 24 13 A199S F227P — L286M S287G A328W Y332G — 1140 25 2614 A199S F227G P285A — S287G A328W Y332G — 3130 27 28 15 A199S F227GP285G — S287G A328W Y332G — 3445 29 30 16 A199S F227G P285Q — S287GA328W Y332G — 2515 31 32 17 A199S F227G P285S — S287G A328W Y332G — 398033 34 18 A199S F227A P285E — S287G A328W Y332G — 2180 35 36 19 A199SF227P P285N — S287G A328W Y332G — 2640 37 38 20 A199S F227S P285A —S287G A328W Y332G — 2450 39 40 21 A199S F227S P285N — S287G A328W Y332G— 2040 41 42 22 A199S F227S — L286M S287G A328W Y332G — 1774 43 44 23A199S F227G — L286M S287G A328W Y332G — 1535 45 46 ^(a)CatalyticEfficiency (k_(cat)/K_(M)) against (—)-cocaine (Approx Fold Increase);The approximate ratio of the k_(cat)/K_(M) value for the BChE mutant tothat for the wild-type BChE against (—)-cocaine.

The terms “polypeptide”, “protein”, and “peptide”, which are usedinterchangeably herein, refer to a polymer of the protein amino acids,or amino acid analogs, regardless of its size or function. Although“protein” is often used in reference to relatively large polypeptides,and “peptide” is often used in reference to small polypeptides, usage ofthese terms in the art overlaps and varies. The term “polypeptide” asused herein refers to peptides, polypeptides, and proteins, unlessotherwise noted. The terms “protein”, “polypeptide”, and “peptide” areused interchangeably herein when referring to a gene product. Thus,exemplary polypeptides include gene products, naturally occurringproteins, homologs, orthologs, paralogs, fragments and otherequivalents, variants, and analogs of the foregoing.

The term “variant” refers to an amino acid sequence that is differentfrom the reference polypeptide by one or more amino acids, e.g., one ormore amino acid substitutions. For example a butyrylcholinesterase(BChE) polypeptide variant differs from wild-type BChE by one or moreamino acid substitutions, i.e., mutations.

The terms “polypeptide fragment” or “fragment”, when used in referenceto a reference polypeptide, refers to a polypeptide in which amino acidresidues are deleted as compared to the reference polypeptide itself,but where the remaining amino acid sequence is usually identical to thecorresponding positions in the reference polypeptide. Such deletions canoccur at the amino-terminus, carboxy-terminus of the referencepolypeptide, or alternatively both. A fragment can also be a “functionalfragment,” in which case the fragment retains some or all of theactivity of the reference polypeptide as described herein. For example,a functional fragment of a particular BChE polypeptide variant retainssome or all of the cocaine hydrolysis activity, i.e., the catalyticefficiency for (−)-cocaine, of the particular BChE polypeptide variant.In this regard, the term “BChE polypeptide variant” is inclusive offunctional fragments of the BChE polypeptide variant. Such fragments aretypically are at least about 300, 325, 350, 375, 400, 425, 450, 475,500, 525, or 550 amino acids long. One or more residues from about 1 to67 and/or one or more residues from about 443 to 574 can be removedwithout substantially affecting the catalytic activity of the BChEpolypeptide variant. As such, the term “BChE polypeptide variant” isinclusive of functional fragments wherein one or more residues from 1 to67 and/or one or more residues from 443 to 574 is truncated relative tothe full-length BChE polypeptide variant. In some embodiments, thefragment includes residues from 1 to 529. See Brimijoin, S. et al.,Neuropsychopharmacology 2008, 33, 2715-2725).

The BChE polypeptide variant (e.g., SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, and 46) canbe formulated in a pharmaceutical composition along with a suitablepharmaceutical carrier known to one skilled in the art.

The present BChE variant polypeptides can be used in treating acocaine-induced condition by administering to an individual, aneffective amount of a BChE variant polypeptides, (e.g., SEQ ID NOS: 2,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, and 46), to lower blood cocaine concentration. The BChEpolypeptide variant can be administered in the form of a pharmaceuticalcomposition in which the BChE polypeptide variant is included with asuitable pharmaceutical carrier. Treatment of a cocaine-inducedcondition using one of the aforementioned BChE polypeptide variants canbe in a manner that will be understood by those skilled in the art.

The preferred dose for administration of a BChE polypeptide variant orpharmaceutical composition in accordance with the presently-describedsubject matter is that amount which will be effective in lowering(−)-cocaine concentration in a patient's bloodstream, and one wouldreadily recognize that this amount will vary greatly depending on thenature of cocaine consumed, e.g., injected or inhaled, and the conditionof a patient. An “effective amount” of butyrylcholinesterase polypeptidevariant or pharmaceutical composition to be used in accordance with thepresently-disclosed subject matter is intended to mean a nontoxic butsufficient amount of the agent, such that the desired prophylactic ortherapeutic effect is produced. Thus, the exact amount of the enzyme ora particular agent that is required will vary from subject to subject,depending on the species, age, and general condition of the subject, theseverity of the condition being treated, the particular carrier oradjuvant being used and its mode of administration, and the like.Similarly, the dosing regimen should also be adjusted to suit theindividual to whom the composition is administered and will once againvary with age, weight, metabolism, etc. of the individual. Accordingly,the “effective amount” of any particular butyrylcholinesterasepolypeptide variant, or pharmaceutical composition thereof, will varybased on the particular circumstances, and an appropriate effectiveamount may be determined in each case of application by one of ordinaryskill in the art using only routine experimentation.

The presently-disclosed subject matter is further illustrated by thefollowing specific but non-limiting examples. The following examples mayinclude compilations of data that are representative of data gathered atvarious times during the course of development and experimentationrelated to the presently-disclosed subject matter.

EXAMPLES

Embodiments of the BChE polypeptide variants of the presently-disclosedsubject matter were made and studied using the following experimentalprocedure.

Site-directed mutagenesis of human BChE cDNA was performed by theQuikChange method of Braman, J.; Papworth, C.; Greener, A. Methods Mol.Biol. 1996, 57, 5731, incorporated herein by this reference. Mutationswere generated from wild-type human BChE in a pRc/CMV expression plasmidin accordance with Xie, W.; Altamirano, C. V.; Bartels, C. F.; Speirs,R. J.; Cashman, J. R.; Lockridge, O. Mol. Pharmacol. 1999, 55, 83, eachof which is incorporated herein by this reference. The expressionplasmid pRc/CMV was kindly provided by Dr. O. Lockridge, University ofNebraska Medical Center (Omaha, Nebr.).

Using plasmid DNA as template and primers with specific base-pairalterations, mutations were made by polymerase chain reaction with PfuDNA polymerase, for replication fidelity. The PCR product was treatedwith Dpn I endonuclease to digest the parental DNA template. Cloned pfuDNA polymerase and Dpn I endonuclease were obtained from Stratagene (LaJolla, Calif.). Modified plasmid DNA was transformed into Escherichiacoli, amplified, and purified. The DNA sequences of the mutants wereconfirmed by DNA sequencing. All oligonucleotides were synthesized bythe Integrated DNA Technologies, Inc. The QIAprep Spin Plasmid MiniprepKit and Qiagen plasmid purification kit and QIAquick PCR purificationkit were obtained from Qiagen (Santa Clarita, Calif.).

BChE mutants were expressed in human embryonic kidney cell line 293T/17.Cells were grown to 80-90% confluence in 6-well dishes and thentransfected by Lipofectamine 2000 complexes of 4 μg plasmid DNA per eachwell. Cells were incubated at 37° C. in a CO₂ incubator for 24 hours andcells were moved to 60-mm culture vessel and cultured for four moredays. The culture medium [10% fetal bovine serum in Dulbecco's modifiedEagle's medium (DMEM)] was harvested for a BChE activity assay.

Human embryonic kidney 293T/17 cells were from ATCC (Manassas, Va.).Dulbecco's modified Eagle's medium (DMEM) was purchased from FisherScientific (Fairlawn, N.J.). Oligonucleotide primers were synthesized bythe Integrated DNA Technologies and Analysis Facility of the Universityof Kentucky. 3,3′,5,5′-Tetramethylbenzidine (TMB) was obtained fromSigma (Saint Louis, Mo.). Anti-butyrylcholinesterase (mouse monoclonalantibody, Product #HAH002-01) was purchased from AntibodyShop (Gentofte,Denmark) and Goat anti-mouse IgG HRP conjugate from Zymed (SanFrancisco, Calif.).

To measure cocaine and benzoic acid, the product of cocaine hydrolysisby BChE, sensitive radiometric assays based on toluene extraction of[³H]-(−)-cocaine labeled on its benzene ring were used in accordancewith Zheng, F.; Yang, W.; Ko, M.-C.; Liu, J.; Cho, H.; Gao, D.; Tong,M.; Tai, H.-H.; Woods, J. H.; Zhan, C.-G. “Most Efficient CocaineHydrolase Designed by Virtual Screening of Transition States”, J. Am.Chem. Soc. 2008, 130, 12148-12155, which is incorporated herein by thisreference. ³H-(−)-cocaine (50 Ci/mmol) was purchased from PerkinElmerLife Sciences (Boston, Mass.).

In brief, to initiate reactions, 100 nCi of [³H]-(−)-cocaine was mixedwith 100 μl of culture medium. Reactions proceeded at room temperature(25° C.) with varying concentrations of (−)-cocaine. Reactions werestopped by adding 300 μl of 0.02 M HCl, which neutralized the liberatedbenzoic acid while ensuring a positive charge on the residual cocaine.[³H]benzoic acid was extracted by 1 ml of toluene and measured byscintillation counting. Finally, the measured (−)-cocaineconcentration-dependent radiometric data were analyzed by using thestandard Michaelis-Menten kinetics so that the catalytic efficiency(k_(cat)/K_(M)) was determined, along with the use of an enzyme-linkedimmunosorbent assay (ELISA) described by Zheng, F.; Yang, W.; Ko, M.-C.;Liu, J.; Cho, H.; Gao, D.; Tong, M.; Tai, H.-H.; Woods, J. H.; Zhan,C.-G. “Most Efficient Cocaine Hydrolase Designed by Virtual Screening ofTransition States”, J. Am. Chem. Soc. 2008, 130, 12148-12155.

The catalytic efficiencies (k_(cat)/K_(M)) of the BChE polypeptidevariants were found to be between about 1000 to about 4000 times thek_(cat)/K_(M) value (9.11×10⁵M⁻¹ min⁻¹) of the wild-type BChE.

Enzyme-linked immunosorbent assays (ELISA) were preformed as follows.The ELISA buffers used were the same as those described in theliterature such as Brock, A.; Mortensen, V.; Loft, A. G. R.;Nergaard-Pedersen, B. J. Clin. Chem. Clin. Biochem. 1990, 28, 221-224;and Khattab, A. D.; Walker, C. H.; Johnston, G.; Siddiqui, M. K.Saphier, P. W. Environmental Toxicology and Chemistry 1994, 13,1661-1667, both of which are incorporated herein by this reference. Thecoating buffer was 0.1 M sodium carbonate/bicarbonate buffer, pH 9.5.The diluent buffer (EIA buffer) was potassium phosphatemonobasic/potassium phosphate monohydrate buffer, pH 7.5, containing0.9% sodium chloride and 0.1% bovine serum albumin. The washing buffer(PBS-T) was 0.01 M potassium phosphate monobasic/potassium phosphatemonohydrate buffer, pH 7.5, containing 0.05% (v/v) Tween-20. All theassays were performed in triplicate. Each well of an ELISA microtiterplate was filled with 100 μl of the mixture buffer consisting of 20 μlculture medium and 80 μl coating buffer. The plate was covered andincubated overnight at 4° C. to allow the antigen to bind to the plate.The solutions were then removed and the wells were washed four timeswith PBS-T. The washed wells were filled with 200 μl diluent buffer andkept shaking for 1.5 h at room temperature (25° C.). After washing withPBS-T for four times, the wells were filled with 100 μl antibody(1:8000) and were incubated for 1.5 h, followed by washing for fourtimes. Then, the wells were filled with 100 μl goat anti-mouse IgG HRPconjugate complex diluted to a final 1:3000 dilution, and were incubatedat room temperature for 1.5 h, followed by washing for four times. Theenzyme reactions were started by addition of 100 μl substrate (TMB)solution. The reactions were stopped after 15 min by the addition of 100μl of 2 M sulfuric acid, and the absorbance was read at 460 nm using aBio-Rad ELISA plate reader.

While the terms used herein are believed to be well understood by one ofordinary skill in the art, the definitions set forth herein are providedto facilitate explanation of the presently-disclosed subject matter.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the presently-disclosed subject matter belongs.Although any methods, devices, and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresently-disclosed subject matter, representative methods, devices, andmaterials are now described.

Following long-standing patent law convention, the terms “a”, “an”, and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a cell” includes aplurality of such cells, and so forth.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as reaction conditions, and so forth usedin the specification and claims are to be understood as being modifiedin all instances by the term “about”. Accordingly, unless indicated tothe contrary, the numerical parameters set forth in this specificationand claims are approximations that can vary depending upon the desiredproperties sought to be obtained by the presently-disclosed subjectmatter.

As used herein, the term “about,” when referring to a value or to anamount of mass, weight, time, volume, concentration or percentage ismeant to encompass variations of in some embodiments ±50%, in someembodiments ±40%, in some embodiments ±30%, in some embodiments ±20%, insome embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%,in some embodiments ±0.5%, and in some embodiments ±0.1% from thespecified amount, as such variations are appropriate to perform thedisclosed method.

As used herein, ranges can be expressed as from “about” one particularvalue, and/or to “about” another particular value. It is also understoodthat there are a number of values disclosed herein, and that each valueis also herein disclosed as “about” that particular value in addition tothe value itself. For example, if the value “10” is disclosed, then“about 10” is also disclosed. It is also understood that each unitbetween two particular units are also disclosed. For example, if 10 and15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

Throughout this document, various references are mentioned. All suchreferences are incorporated herein by reference.

The invention claimed is:
 1. An isolated butyrylcholinesterasepolypeptide variant comprising the amino acid sequence of SEQ ID NO: 34;or the amino acid sequence encoded by a nucleic acid sequence of SEQ IDNO:
 33. 2. A pharmaceutical composition comprising: abutyrylcholinesterase polypeptide variant of claim 1; and a suitablepharmaceutical carrier.
 3. A method of treating a cocaine-inducedcondition comprising administering to an individual an effective amountof a pharmaceutical composition of claim 2 to lower blood cocaineconcentration.
 4. A method of treating a cocaine-induced conditioncomprising administering to an individual an effective amount of abutyrylcholinesterase polypeptide variant of claim 1 to lower bloodcocaine concentration.
 5. The method of claim 4, wherein saidbutyrylcholinesterase polypeptide variant exhibits a one-hundred-fold ormore increase in cocaine hydrolysis catalytic efficiency compared tobutyrylcholinesterase.